L-cysteine (Cys) is one of the main amino acid and its deficiency could cause several diseases and biological disorders in our body. Thus, it is important to accurately detect Cys level in biological samples. Herein, we reported a facile strategy to fabricate novel electrode material consisting of conductive hybrid film made of copper oxide-boron nitride nanosheets (CuO/BN). Powdered X-ray diffraction (PXRD) and Fourier transform infrared (FT-IR) spectroscopy results confirmed the successful formation of CuO/BN hybrid nanocomposite. Next, electrochemical and electro-catalytic properties of CuO/BN hybrid film is studied by cyclic voltammetry and amperometry. Interestingly, CuO/BN coated glassy carbon electrode (GCE) showed excellent electro-catalytic activity towards Cys oxidation in phosphate buffer solution (0.1 M PBS). Newly developed CuO/BN film modified electrode is exhibited high oxidation peak current at lower potential of + 0.45 V compared to bare GCE. CuO/BN/GCE sensor is also utilized for the detection of Cys from 1 to 10 μM and limit of detection (LOD) was found to be 0.58 μM. The selectivity of the sensor is determined by detecting 5 μM Cys in the presence of different interferent molecules. Furthermore, repeatability, reproducibility and stability of the CuO/BN/GCE sensor are also investigated. Finally, CuO/BN/GCE sensor is successfully applied to detect spiked Cys in human blood serum samples with good recovery.